Project Abstract DDX3X is an essential translation initiation factor that is conserved from yeast to human. Deregulated DDX3X expression, DDX3X mutations, or both, are linked to several cancers, including breast, colorectal, lung, oral squamous cell cancers, and medulloblastoma, a pediatric brain cancer. In medulloblastoma, DDX3X mutations promote formation of cytoplasmic messenger ribonucleoprotein (mRNP) granules (stress granules), which form by liquid-liquid phase separation and are associated with global changes in translation. Several lines of evidence suggest that stress granule formation and global changes in translation occur in other cancers as well, and that DDX3X plays a key role in linking these processes. How DDX3X coordinates translation initiation and granule formation is not understood. This proposal directly addresses this problem. Aim 1 combines enzymological techniques with fluorescence microscopy to define the relationship between DDX3X phase separation and helicase activity. Preliminary data show that direct interaction with the vaccinia virus protein K7, which binds the N-terminal intrinsically disordered region of DDX3X, disrupts DDX3X granules and suppresses DDX3X enzymatic activity. The effect of RNA, ATP, and K7 binding on DDX3X granule dynamics will be characterized to develop a model for the impact of phase separation on DDX3X helicase activity and to test the ability of K7 to disrupt cancer-associated DDX3X granules. Aim 2 uses ribosome profiling to characterize the interplay between DDX3X phase separation and translational control. Translational signatures resulting from DDX3X depletion, DDX3X granule formation, and the disruption of DDX3X granules by K7 will be compared to identify how DDX3X phase separation alters translation and whether these changes can be reversed by granule disruption. Expected data will chart new territory for understanding biochemical and enzymatic aspects of mRNP granule formation. The work will also provide a roadmap for the mechanistic analysis of other RNA binding proteins that associate with cytoplasmic granules. Finally, since inhibition of DDX3X catalytic activity promotes granule formation that may drive tumorigenesis, as is suggested for medulloblastoma, the simultaneous disruption of granules and suppression of DDX3X activity by K7 or similar effectors may represent a new strategy for the treatment of cancers linked to DDX3X malfunction.